Methods of screening nucleic acids for nucleotide variations

ABSTRACT

The present invention provides a method of detecting nucleotide variation within a first nucleic acid, comprising generating a set of single-stranded extension products from a first nucleic acid in the presence of modified nucleotide bases, wherein the extension products incorporate modified nucleotides and thereby limit exonuclease activity to the 3′-terminal nucleotide base, and wherein the extension products have variable lengths, hybridizing the variable length extension products to a reference nucleic acid, contacting the hybridizing nucleic acids with an enzyme which can remove and replace the 3′-terminal nucleotide of the extension products in the presence of selected labeled nucleotides, wherein extension products that terminate with a 3′-nucleotide that does not hybridize with the corresponding position on the reference nucleic acid are replaced with one or more nucleotides that hybridize with the corresponding nucleotides on the reference nucleic acid and wherein those extension products that had a non-hybridizing nucleotide at the 3′-terminus can now be distinguished from those extension products that had a hybridizing nucleotide at the 3′-terminus, and distinguishing those extension products that had a non-hybridizing nucleotide at their 3′-terminus from those extension products that had a hybridizing nucleotide at their 3′-terminus, thereby detecting nucleotide variation in the first nucleic acid. Alternatives of this method are also provided which can also detect mutations in a nucleic acid at the penultimate 3′-terminal position on the single-stranded extension products.

This application is a continuation-in-part of Ser. No. 90/137,075 filedAug. 20, 1998 now U.S. Pat. No. 6,150,105.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the field of detecting nucleotide variationsin a nucleic acid. More particularly, the invention relates to methodsof detecting nucleotide variations in a nucleic acid by generatingvariable length copies of a nucleic acid from a sample, hybridizingthose generated nucleic acids to reference nucleic acids, and detectingthe presence or absence of nucleotide variations at the 3′-terminalposition or the penultimate 3′-position on the variable length nucleicacids.

2. Background Art

The number of diseases that are linked to gene mutations continues toincrease as the sequence of the human genome is unraveled. Nucleic acidsequencing is the ultimate standard for detecting nucleotide variations.Nucleic acid sequencing is well suited for detecting unknown mutationsor polymorphisms that may occur at any base within a target nucleic acidsegment. The chemistry of enzymatic DNA sequencing, the most commonlyused method, has essentially remained the same since its conception(Sanger et al., Proc. Natl. Acad. Sci. U.S.A., 74, 5463 (1977)). The arthas been improved by technology that has allowed for its automation suchas the introduction of fluorescent dyes, robotics and improvedelectrophoretic systems with automated detection. However, if geneticvariations occur at a low frequency in the sample population, automationcomes at a cost that is too high for most laboratories. Even in a manualmode, sequencing can be cost prohibitive because it is labor intensive.Thus, there is a need in the art for a simple inexpensive process toscreen nucleic acids for unknown nucleotide variations prior tosequencing.

That need in the art is evident by the number of methods being developedto screen for unknown mutations. Single strand conformation polymorphism(SSCP) detects mutations in an unknown sample by comparing its migrationrate in a single stranded state to a known sample in a non-denaturinggel, as disclosed by Orita et al., Genomics, 5:874-879 (1989). Changesin nucleotide sequence affect the secondary structure or conformation ofa DNA molecule which may alter its migration rate duringelectrophoresis. This technique, however, is limited to small targetsless than 200 bp, has limited sensitivity, and requires rigidelectrophoresis conditions to be reproducible. Improvements in SSCPanalysis such as dideoxy fingerprinting, both unidirectional (Sarkar etal., Genomics, 13:441-443 (1992)) and bidirectional (Liu et al., Hum.Mol. Genet., 5:107-114 (1996)), and restriction endonucleasefingerprinting (Liu and Sommer, Biotechniques, 18:470-477 (1995)) candetect mutations over a 1 kb span but sacrifice sensitivity forsimplicity since the complex pattern of DNA bands generated by theseprocesses makes it difficult to readily detect mutations.

Another method that is used for screening for nucleotide variations in anucleic acid is based on the differential mobility of heteroduplexmolecules as they migrate through a gel matrix. In its simplest formcalled heteroduplex analysis, an uncharacterized DNA segment, usually anamplification or PCR product, is mixed with the corresponding wild typesegment, heated, and allowed to slowly renature, as first described byNagamine et al. (Am. J. Hum. Genet., 45, 337-339 (1989)). If theuncharacterized nucleic acid has a different sequence than the wild typesequence, heteroduplex molecules are formed. Base mismatches in theheteroduplex alter its migration rate allowing it to be partiallyresolved from the homoduplex in a non-denaturing gel.

A more sensitive approach called denaturing gradient gel electrophoresis(DGGE) subjects heteroduplex molecules to increasing levels ofdenaturant in a gradient gel format, as first described by Fisher andLerman. (Proc. Natl. Acad. Sci. U.S.A., 80:1579-1583 (1983)). As theheteroduplex molecules migrate through the denaturant, they begin tomelt, or denature. At this point migration is slowed and is no longerlinear. The melting point is slightly different for homoduplexmolecules, allowing partial resolution of heteroduplex molecules.Precise control of field strength, temperature and time are critical toachieving reproducible results, and difficult to consistently reproduce.

With constant denaturing gel electrophoresis (CDGE), these variables areless critical since the concentration of denaturant is the samethroughout the gel (Hovig et al., Mut. Res., 262:63-71 (1991)). Asignificant limitation of this technique is that a nucleic acid segmentmay have more than one melting domain for which separate gels atdifferent denaturant concentrations must be run.

Temporal temperature gradient gel electrophoresis (TTGE) seeks tocircumvent this problem by gradually increasing the temperature duringelectrophoresis, as described by Borresen et al. (Bioradiations,99:12-113 (1997)). This is a hybrid technique between CDGE andtemperature gradient gel electrophoresis which uses temperature only asa denaturant (Rosenbaum and Riesner, Biophys. Chem., 26:235-246 (1987)).As expected, however, this technique is also difficult to perform andalso difficult to reproduce.

A recently introduced technique called base excision sequence scanning(BESS) improves upon dideoxy fingerprinting with ddTTP by obviating theneed for a separate sequencing reaction (Epicentre Technologies,Madison, Wiss.). The target of interest is amplified by PCR using alabeled primer and a limiting amount of dUTP. After amplification, theproducts are treated with uracil DNA glycosylase to cleave at uracilsites. Denaturing gel electrophoresis of the fragments then produces aladder almost identical to a dideoxy T sequencing ladder. The techniqueis useful for screening DNA segments up to 1 kb for mutations, but islimited by the resolution of gel electrophoresis and it does not detectG to C transversions or vice versa.

Another recently introduced technique uses a structure specificendonuclease called cleavase to digest intrastrand structures andproduce fragment length polymorphisms (CFLP) and is described by Brow etal., J. Clin. Microbiol., 34:3129-3137 (1996). The structures arecreated by denaturing a segment of DNA and then quickly cooling it tothe digestion temperature and adding the enzyme. The folding pattern fora given segment may be altered by sequence variations that upondigestion with the enzyme produces a unique banding pattern on adenaturing gel. This technique, however, is severely limited by theresolution of the gel electrophoresis and the complex pattern of DNAbands generated by the process which makes it difficult to detectmutations.

Detection of mutations by chemical or enzymatic cleavage of base pairmismatches in heteroduplex DNA has been described by Noack et al., Proc.Natl. Acad. Sci. U.S.A., 83:586-590 (1986), Cotton et al. Proc. Natl.Acad. Sci. U.S.A., 85:4394-4401 (1988), Cotton et al., U.S. Pat. No.5,202,231, (Winter et al., Proc. Natl. Acad. Sci. U.S.A., 82:7575-7579(1989), Myers et al., Science, 230:1245-1246 (1985)), (Lu and Hsu,Genomics, 14:249-255 (1992),) and U.S. Pat. No. 5,698,400. Many of thesetechniques are limited by the inability of the cleavage reagents torecognize all types of base pair mismatches, and for others this can beovercome by analyzing both strands of a DNA segment. To date, widespreaduse of these techniques has not been observed, partly because theyrequire highly toxic reagents and the procedures are difficult toperform.

The miniaturization of the DNA hybridization process onto a small solidsurface, known as a DNA chip or micro array, allows the analysis of DNAsegments without gel electrophoresis. See Macevicz, U.S. Pat. No.5,002,867, Drmanac., U.S. Pat. No. 5,202,231, Lipshutz et al.,Biotechniques, 9(3):442-447 (1995) and Chee et al., Science, 274:610-614(1996). The resolution of gel electrophoresis, however, strictly limitsthe size of the DNA segment that can be analyzed for all of theaforementioned mutation detection technologies including DNA sequencingand the high cost of the equipment,and chips used in this process limitits wide spread use.

The present invention provides needed improvements over these prior artmethods by providing methods which can detect all possible basevariations including single and multiple base substitutions, insertionsand deletions. These variations may occur at one or more sites andaffect one or more nucleotides at each site for a given locus. Secondly,as a screening process, these methods provide a clear positive ornegative result. Thirdly, the process is not limited by the resolutionpower of gel electrophoresis and therefore allowing the analysis of DNAsegments greater that 1 kb in size. Lastly, by way of eliminatingelectrophoretic detection, it is highly amenable to automation andtherefore suitable for high volume screening.

SUMMARY OF THE INVENTION

In accordance with the purpose(s) of this invention, as embodied andbroadly described herein, this invention, in one aspect, relates to amethod of detecting nucleotide variation within a first nucleic acid,comprising generating a set of single-stranded extension products from afirst nucleic acid in the presence of modified nucleotide bases, whereinthe extension products incorporate modified nucleotides and therebylimit exonuclease activity to the 3′-terminal nucleotide base, andwherein the extension products have variable lengths, hybridizing thevariable length extension products to a reference nucleic acid,contacting the hybridizing nucleic acids with an enzyme which can removeand replace the 3′-terminal nucleotide of the extension products in thepresence of selected labeled nucleotides, wherein extension productsthat terminate with a 3′-nucleotide that does not hybridize with thecorresponding position on the reference nucleic acid are replaced with anucleotide that hybridizes with the corresponding nucleotide on thereference nucleic acid and wherein those extension products that had anon-hybridizing nucleotide at the 3′-terminus can now be distinguishedfrom those extension products that had a hybridizing nucleotide at the3′-terminus, distinguishing those extension products that had anon-hybridizing nucleotide at their 3′-terminus from those extensionproducts that had a hybridizing nucleotide at their 3′-terminus, therebydetecting nucleotide variation in the first nucleic acid.

The invention also provides a method of detecting within a first nucleicacid the presence of a nucleotide variation, comprising generating a setof single-stranded extension products in the presence of modifiednucleotide bases, wherein the extension products incorporate modifiednucleotides that limit exonuclease activity to the 3′-terminalnucleotide base, and wherein the extension products have variablelengths, hybridizing the variable length extension products to areference nucleic acid, contacting the hybridizing nucleic acids with anenzyme which can remove and replace the 3′-terminal nucleotide of theextension products in the presence of a selected modified nucleotidewhich is resistant to further replacement and when incorporated into anextension product inhibits further extension of the extension product,wherein extension products that terminate with a non-modified nucleotidecan be further extended and thereby distinguished from those extensionproducts that cannot be further extended, removing the unincorporatedselected modified nucleotide, extending those extension products thatcan be further extended, distinguishing those extension products thatare further extended from those extension products that cannot befurther extended, thereby detecting nucleotide variation in the firstnucleic acid.

The invention further provides a method of detecting nucleotidevariation within a first nucleic acid, comprising generating a set ofsingle-stranded extension products in the presence of modifiednucleotide bases and chain-terminating nucleotide bases from a firstnucleic acid, wherein the extension products incorporate modifiednucleotides that limit exonuclease activity to the 3′-terminalnucleotide base, and wherein the extension products have variablelengths, hybridizing the variable length extension products to areference nucleic acid, contacting the hybridizing nucleic acids with anenzyme which can remove and replace the 3′-terminal nucleotide of theextension products in the presence of deoxynucleotide triphosphates,wherein the penultimate 3′-nucleotide is resistant to removal from theextension products, whereby extension products containing a penultimate3′-nucleotide that does not hybridize with the corresponding position onthe reference nucleic acid is not replaced with a nucleotide thathybridizes with the corresponding nucleotide on the reference nucleicacid and thereby cannot be further extended, extending those extensionproducts that can be further extended, distinguishing those extensionproducts that are further extended from those extension products thatcannot be further extended, thereby detecting nucleotide variation inthe first nucleic acid.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows a schematic for the single-base extension template exchangereaction extension detection method.

FIG. 2 shows a schematic for the extension detection method.

FIG. 3 shows a schematic for the non-extension template exchangeextension reaction detection method using exo-resistant termini.

FIG. 4 shows a schematic for the non-extension template exchangeextension reaction detection method using dideoxynucleotidesincorporated at the 3′-termini.

FIG. 5 is a diagram of an example of the methods of the inventionadapted to a microtiter plate format.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of the preferred embodiments of theinvention and the Examples included therein.

Before the present compounds and methods are disclosed and described, itis to be understood that this invention is not limited to specificnucleic acids, chain terminating nucleotides, editing enzymes, extensionand/or amplification enzymes, detectable moieties, and other reagentsused in the methods described herein, as such may, of course, vary. Itis also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a nucleic acid” includes multiple copies of the nucleicacid and can also include more than one particular species of molecule.

In one aspect, the invention relates to a method of detecting nucleotidevariation within a first nucleic acid, comprising generating a set ofsingle-stranded extension products from a first nucleic acid in thepresence of modified nucleotide bases, wherein the extension productsincorporate modified nucleotides and thereby limit exonuclease activityto the 3′-terminal nucleotide base, and wherein the extension productshave variable lengths, hybridizing the variable length extensionproducts to a reference nucleic acid, contacting the hybridizing nucleicacids with an enzyme which can remove and replace the 3′-terminalnucleotide of the extension products in the presence of selected labelednucleotides, wherein extension products that terminate with a3′-nucleotide that does not hybridize with the corresponding position onthe reference nucleic acid are replaced with a nucleotide thathybridizes with the corresponding nucleotide on the reference nucleicacid and wherein those extension products that had a non-hybridizingnucleotide at the 3′-terminus can now be distinguished from thoseextension products that had a hybridizing nucleotide at the 3′-terminus,distinguishing those extension products that had a non-hybridizingnucleotide at their 3′-terminus from those extension products that had ahybridizing nucleotide at their 3′-terminus, thereby detectingnucleotide variation in the first nucleic acid.

Nucleotide variation as used herein refers to any nucleotidesubstitution at one or more positions in a nucleic acid (the firstnucleic acid) and any insertion or deletion at one or more positions ona nucleic acid, and any combination thereof. For example, a nucleic acidcan have a single base substitution in a region that is being assayed,or that nucleic acid can have multiple base substitutions in the region,or any combination of base substitutions, insertions, and deletions.Nucleotide variation as used herein also refers to any nucleotidemodification that could give rise to an altered phenotype or genotype.The methods described herein can detect the presence of a basesubstitution, a deletion, and an insertion, and any other mutation thatcauses a nucleotide on a nucleic acid to not hybridize with thecorresponding nucleotide on a separate, at least partially complementarynucleic acid.

The first nucleic acid can be a single stranded or double strandednucleic acid from a sample, i.e. a patient or experimental sample, andthe reference nucleic acid can be a standard or reference nucleic acidto which the first nucleic acid is hybridized, and/or compared. In themethods described herein, one or more strands of the first nucleic acidare used to generate a set of single-stranded extension products thatare at least partially resistant to 3′→5′ exonuclease activity and canoptionally contain a chain-terminating nucleotide or deoxynucleotide attheir 3′-terminus, to generate a set of single-stranded extensionproducts of variable length.

The generation or isolation of the first nucleic acid for use in theinvention can be optionally facilitated by amplification of the targetregion by cloning the region of interest into a replication vector suchas a plasmid or a phage, to generate either double or single strandedmolecules. If the first nucleic acid is generated by cloning the nucleicacid, there are many known techniques for isolating the nucleic acidfrom the cloning vector after the nucleic acid within the cloning vectorhas been replicated, such as phage isolation, plasmid isolation vialysis of bacteria followed by denaturation of the cellular proteins andcentrifugation of the nucleic acids, or even density banding of plasmidor phage nucleic acids by density gradient centrifugation.

One skilled in the art will recognize that there are many other knownamplification techniques for generating copies of one or more strands ofa nucleic acid, such as the polymerase chain reaction (PCR). Otheramplification techniques can also be used to amplify a nucleic acid,however, the self-sustained sequence replication (3SR) system, thetranscription-based amplification system (TAS), and the RNA replicationsystem based on Qβ replicase. The product of the amplification, thefirst nucleic acid, and/or the reference nucleic acid can therefore be aDNA or an RNA, either single stranded or double stranded. If the productof the amplification is double stranded, single strands can be isolatedby methods well known in the art, such as the use of biotin-linkedamplification primers or the selective degradation of a phosphorylatedstrand using lambda exonuclease as described by Higuchi and Ochman,Nucleic Acids Research, 17:5865 (1989). Alternative methods for theproduction of single stranded templates are also known in the art suchas asymmetric PCR (Ausbel et al., “Current Protocols in MolecularBiology”, John Wiley & Sons, New York (1987)) and solid phase capture(Holtman et al., Nucleic Acids Research, 17:4937-4946 (1989)). If theproduct of the amplification procedure is an RNA, DNA can be generatedfrom that RNA using techniques well known in the art such as the use ofreverse transcriptase.

Whether the first nucleic acid for use in the methods described hereinhas been amplified or whether the first nucleic acid is obtaineddirectly, such as from a patient sample or any other experimentalsample, that nucleic acid is then used as a template for generating aset of single-stranded extension products of variable length. Forexample, a chain-terminating dideoxynucleotide can be used in thereaction such that the resultant set of single-stranded extensionproducts contains single-stranded extension products that terminate ateach position where the dideoxynucleotide is incorporated. Preferably,the set of variable-length single-stranded extension products in thisembodiment would contain at least one single-stranded extension productthat incorporated a dideoxynucleotide into every available position forthat particular species of dideoxynucleotide; i.e. a “sequencingladder.” Generation of dideoxy sequencing ladders is well known in theart and may be accomplished with commercially available sequencing kits.In a preferred embodiment of the present invention, the sequencingladder is generated by thermal cycle sequencing to generate a pluralityof sequencing ladders corresponding to the first nucleic acid tofacilitate later detection of those single-stranded extension productsthat have 3′-terminal nucleotide that hybridizes to a nucleotide at thecorresponding position on a reference nucleic acid or a 3′-terminalnucleotide that does not hybridize to a nucleotide at the correspondingposition on a reference nucleic acid.

Alternatively, the set of single-stranded extension products of variablelength can be generated by different procedures. For example, thesingle-stranded extension products can be generated by extension of theprimer sequence without chain-terminating nucleotides present in thereaction mixture, but where the deoxynucleotides are present in alimiting amount. Alternatively, the deoxynucleotides can be present in asufficient amount to synthesize a set of single-stranded extensionproducts that correspond to the total length of the first nucleic acid,but which are then used to generate a set of variable lengthsingle-stranded extension products through partial terminal degradationof the single-stranded extension products, for example, usingexonuclease I or III, or T4 DNA Polymerase in the absence of or alimiting amount of deoxynucleotides. One skilled in the art willappreciate that the particular technique or techniques used to generatethe set of single-stranded extension products can vary.

One skilled in the art will also recognize that the generation of theset of single-stranded extension products will require a primer for theextension reaction to proceed. The primer can be internal, such ashybridization of a 3′-region to the 5′-region of the same molecule, orthe primer can be external, such as the use of a synthesized primer thatcan hybridize to a preferred site or sites on the first nucleic acid.

The first nucleic acid, the single-stranded extension products, and theprimer can be modified such as by being linked to another molecule suchas biotin, digoxigenin, a hapten, an antibody, an enzyme, or anothermoiety that can facilitate the isolation and/or detection of themolecules. As discussed in the Example section herein, a primer can belinked to biotin, and the set of single-stranded extension productsproduced as a result of extension reactions using that modified primercan be isolated from the first nucleic acid and the other components ofthe extension reaction using streptavidin-coated magnetic beads.

Optionally, the modified first nucleic acid, the single-strandedextension products, and/or the primer can be purified or isolated from areaction mixture and/or other nucleic acids by binding them to a solidsupport. For example, a nucleic acid complementary to at least a portionof a primer used in the extension reaction can be linked to a solidsupport, such as beads used in column chromatography, and after theprimer has been extended, that reaction mixture can be heated todenature the extension products away from the first nucleic acid, andthat mixture passed through the column under conditions such that theextension products hybridize to a nucleic acid linked to the beads andnot to the first nucleic acid, and whereby the extension products atethereby purified from the first nucleic acid. For example, the primercan comprise the sequence complementary to the first nucleic acid andalso have a 5′ region comprising a sequence not complementary to thefirst nucleic acid, such as a poly(C) region. The nucleic acid linked tothe beads can then comprise a poly(G) region that can hybridize to thepoly(C) region under conditions such that the extension product does nothybridize to the first nucleic acid. One skilled in the art willrecognize that there are many other examples of procedures used toseparate one strand of an extension reaction from its complementary, orpartially complementary strand, and the methods described herein are notlimited to any specific isolation, separation, or purificationprocedure.

Alternatively, a primer used to generate the set of single-strandedextension products can be phosphorylated at the 5′ end to allowsubsequent degradation of the phosphorylated strand with lambdaexonuclease as described by Higuchi and Ochman (Nucleic Acids Research,17:5865 (1989)). Alternative methods for the production of singlestranded templates are also known in the art such as asymmetric PCR(Ausbel, et al., “Current Protocols in Molecular Biology”, John Wiley &Sons, New York (1987)) and solid phase capture (Holtman et al., NucleicAcids Research, 17:4937-4946 (1989)).

The single-stranded extension products are typically generated in thepresence of at least one type of modified nucleotide is used in thegeneration reaction and thereby incorporated in the single-strandedextension products, such that the single-stranded extension products areat least partially resistant to 3′→5′ exonuclease activity. The modifiednucleotides main function after incorporation as a nucleotide is tolimit subsequent 3′→5′ exonuclease activity to the 3′ dideoxynucleotide.These modified nucleotides are well known in the art and include, butare not limited to, thio-modified deoxynucleotide triphosphates andborano-modified deoxynucleotide triphosphates (Eckstein and Gish, Trendsin Biochem. Sci., 14:97-100 (1989) and Porter Nucleic Acids Research,25:1611-1617 (1997)). Boronated nucleotides have been shown to be morereadily integrated in an extension reaction by a thermostable polymerasethan are thio-modified dNTPs, and have a greater resistance to nucleases(Porter et al., Nucleic Acids res. 25(8):1611-1617 (1997).

After the set of single-stranded extension products of variable lengthhas been generated, and optionally isolated from the first nucleic acid,the single-stranded extension products are then hybridized to areference nucleic acid. This reference nucleic acid can be a nucleicacid that is typically considered “wild-type” for a particular gene orportion of a gene including structural and regulatory regions of thegene. For example, the reference nucleic acid can be a nucleic acid thatis known to be a locus for mutations in or near a particular gene thatwhen mutated, typically gives rise to an altered phenotype or disease inan individual. Alternatively, the mutations can result in a differentphenotype that is considered beneficial, such as a bacterial species nowbeing able to detoxify a toxin. Any mutation is contemplated as beingdetected by the methods described herein, and the specific identity ofthe mutation does not limit the applicability of these methods.Additionally, the reference nucleic acid can be isolated, generated,synthesized, or amplified for use in the methods described herein by anymethods described in the art since the source of the reference nucleicacid is also not limiting to the present methods.

The precise conditions of the hybridizations will, of course, varydepending on the specific sequence of the reference nucleic acid and thefirst nucleic acid. The specific conditions are readily obtainable byone skilled in the art, and typical hybridization conditions andoptimization conditions are available from a wide variety of sourcereferences. For example, Innis et al. (“PCR Protocols: A Guide toMethods and Applications” Academic Press, Inc. 1990) and Erlich, H. A.(PCR Technology, Principles and Applications for DNA Amplification) bothdisclose standard hybridization conditions for nucleic acidamplification, and Sambrook et al. (“Molecular Cloning, a LaboratoryManual” Cold Spring Harbor Laboratory Press (1989)) set forth generalmethods for typical nucleic acid hybridizations and optimizationprocedures for those methods. Optionally, the specific hybridizationcondition for hybridization between the single-stranded extensionproducts and the reference nucleic acids is such that activity of anenzyme which can remove and replace the 3′-terminal nucleotide of thesingle-stranded extension product is retained, and typically, highlyactive and specific.

After the single-stranded extension products have been hybridized to thereference nucleic acids, an enzyme that can remove and replace the3′-terminal nucleotide of the extension products (i.e. a “proofreading”enzyme) is added to these hybrids. In one embodiment of the presentinvention, this reaction can take place in the presence of achain-terminating nucleotide and if the 3′-terminal nucleotidehybridizes to the reference nucleic acid, that 3′-terminal nucleotidewill be replaced with a chain-terminating nucleotide of the sameidentity (FIG. 1). Where the 3′-terminal nucleotide of the extensionproduct does not hybridize to the reference nucleic acid, such as wherethe 3′-terminal nucleotide of the extension represents a mutation in thefirst nucleic acid at that particular position, the proofreading enzymewill remove this mismatched base on the extension product and replace itwith a base that hybridizes to the reference nucleic acid, and which canbe detected.

The specific proofreading enzyme used in the methods described herein isnot limited to a DNA polymerase, but includes any enzyme, or combinationof enzymes which can remove a 3′-terminal nucleotide and can replacethat 3′-terminal nucleotide with another nucleotide of the same ordifferent identity. For example, the proofreading enzyme can be, forexample, a thermostable polymerase such as Vent® DNA Polymerase, DeepVent® DNA Polymerase, E. coli DNA Polymerase I, Klenow Fragment DNAPolymerase I, T4 DNA Polymerase, T7 DNA Polymerase, Ultima® DNAPolymerase, and Pfu® DNA Polymerase. Alternatively, the enzyme thatremoves the 3′-terminal nucleotide can be an exonuclease such as E. coliexonuclease III or exonuclease I used in combination with a polymerasesuch as T4 DNA Polymerase, to effectively achieve the same result withan additional step. Alternatively, a non-proofreading polymerase enzymecan be used in the methods described herein. The inability of anon-proofreading polymerase to extend a mismatch is well known in theart and is the basis of allele specific PCR and amplification refractorymutation systems (ARMS). See Wu et al., Proc. Natl Acad. Sci. U.S.A.,86:2757-2760 (1989) and Newton et al., Nucleic Acids Res., 17:2503-2516(1989).

Thus, in one embodiment, the invention provides a method of detecting anucleotide variation within a first nucleic acid, comprising generatinga set of single-stranded extension products from the first nucleic acidin the presence of modified nucleotide bases, wherein the extensionproducts incorporate modified nucleotides and thereby limit exonucleaseactivity to the 3′-terminal nucleotide base, and wherein the extensionproducts have variable lengths; hybridizing the variable lengthextension products to a reference nucleic acid; contacting thehybridizing nucleic acids with a first enzyme which can remove the3′-terminal nucleotide of the extension products, and with a secondenzyme which extends those extension products that can be furtherextended in the presence of selected labeled nucleotides, whereinextension products that terminate with a 3′-nucleotide that does nothybridize with the corresponding position on the reference nucleic acidare replaced with a nucleotide that hybridizes with the correspondingnucleotide on the reference nucleic acid and wherein those extensionproducts that had a non-hybridizing nucleotide at the 3′-terminus cannow be distinguished from those extension products that had ahybridizing nucleotide at the 3′-terminus; and distinguishing thoseextension products that had a non-hybridizing nucleotide at their3′-terminus from those extension products that had a hybridizingnucleotide at their 3′-terminus, thereby detecting nucleotide variationin the first nucleic acid. The hybridizing nucleic acids may either becontacted sequentially with the first enzyme followed by the secondenzyme, or contacted simultaneously with the first enzyme and the secondenzyme.

The nucleotide that is incorporated into the extension product that hada 3′-terminal nucleotide that did not hybridize to the reference nucleicacid can comprise a nucleotide that can be detected in the presence ofnucleotides that have the same identity as the 3′-terminal nucleotide ofthe extension products where the 3′-terminal nucleotide initiallyhybridized to the reference nucleic acid. For example, where theextension reaction is performed in the presence of a chain-terminatingnucleotide corresponding to dATP, the excision/replacement reactionwhere the 3′-terminal nucleotide that does not hybridize to thereference nucleic acid is replaced by a nucleotide that does hybridizeto the reference nucleic acid can be performed in the presence ofradiolabeled dGTP, dCTP, and dTTP. In this example, where any of theradiolabeled dGTP, dCTP, and dTTP nucleotides are incorporated into theextension product, that product can be detected by the presence ofradioactivity. Further, the reaction can contain an internal control,such as a differently labeled nucleotide, such as ³⁵S-dATP orfluorescent dATP in this example, whereby that label can be incorporatedinto the single-stranded extension product that has a 3′-nucleotide thathybridized to the corresponding position on the reference nucleic acid,so one can monitor the reaction for activity, especially in the eventthat few or no mutations are detected, since both those extensionproducts that had a hybridizing nucleotide at their 3′-terminalnucleotide and those that had a non-hybridizing nucleotide at the3′-terminal nucleotide can be detected, and also distinguished.Alternatively, the label can be only incorporated into the extensionproduct that had a hybridizing nucleotide at its 3′-terminal nucleotideposition.

The specific label that one uses to detect the presence of a mutationcan, of course, vary. For example, the label can comprise a radiolabel,a fluorescent label, a luminescent label, an antibody linked to anucleotide that can be subsequently detected, a hapten linked to anucleotide that can be subsequently detected, or any other nucleotide ormodified nucleotide that can be detected either directly or indirectly.Therefore the specific method or methods used to distinguish between thesingle-stranded extension products that had a 3′-terminal nucleotidethat hybridized to the reference nucleic acid and the single-strandedextension products that had a 3′-terminal nucleotide that did nothybridize to the reference nucleic acid will vary depending upon thespecific label that is used in the methods described herein. Forexample, if the labeled nucleotide is a radiolabeled nucleotide, thedetection method can comprise scintillation counting or exposing thereaction products to film, which when developed, can distinguish betweenthe labeled nucleic acids and the unlabeled nucleic acids.

Also provided by the present invention is a method of detecting within afirst nucleic acid the presence of a nucleotide variation, comprisinggenerating a set of single-stranded extension products from the firstnucleic acid in the presence of modified nucleotide bases, wherein theextension products incorporate modified nucleotides that limitexonuclease activity to the 3′-terminal nucleotide base, and wherein theextension products have variable lengths, hybridizing the variablelength extension products to a reference nucleic acid, contacting thehybridizing nucleic acids with an enzyme which can remove and replacethe 3′-terminal nucleotide of the extension products in the presence ofa selected modified nucleotide which is resistant to further replacementand when incorporated into an extension product inhibits furtherextension of the extension product, wherein extension products thatterminate with a non-modified nucleotide can be further extended andthereby distinguished from those extension products that cannot befurther extended, removing the unincorporated selected modifiednucleotide, extending those extension products that can be furtherextended, distinguishing those extension products that are furtherextended from those extension products that cannot be further extended,thereby detecting nucleotide variation in the first nucleic acid.

This particular method is based on the ability of a proofreadingpolymerase to replace a matching terminal chain-terminating nucleotidesuch as a dideoxynucleotide with a modified chain-terminating nucleotidethat is resistant to 3′→5′ exonuclease activity in an irreversible or“suicide” reaction. Single-stranded extension products of variablelength generated from a first nucleic acid are mixed with a knowntemplate or a reference nucleic acid to form a stable hybrid. The hybridis exposed to a proofreading polymerase, or in an equivalent reactionthereof as discussed above, in the presence of a chain terminatingnucleotide that is resistant to further 3′→5′ exonuclease activity, suchas a α-thio-dideoxynucleotide, that is the base-equivalent of the 3′base of the extension products, under conditions suitable for excisionof the 3′-terminal nucleotide of the extension product and itsreplacement with the chain terminating nucleotide that is resistant tofurther 3′→5′ exonuclease activity (FIG. 2). If the 3′-terminalnucleotide of the extension product hybridizes to the correspondingposition on the first nucleic acid, then that 3′-terminal nucleotide isexcised and replaced with a corresponding chain terminating nucleotidethat is resistant to further 3′→5′ exonuclease activity. Theproofreading polymerase enzyme can no longer proofread the 3′-terminalnucleotide and also cannot extend the matching terminal base because itis a chain-terminating nucleotide. If, however, the 3′-terminalnucleotide of the single-stranded extension product does not hybridizeto the reference nucleic acid (i.e., there is a mismatch at the terminalbase), the proofreading polymerase will excise the 3′-terminalnucleotide of the single-stranded extension product but will not inserta chain-terminating nucleotide into that position, because, for example,a chain-terminating nucleotide which can hybridize to the correspondingposition on the reference nucleic acid is not present in the reactionmixture. That proofread extension product can then be further extendedand that extended product detected. Thus, extension products whose3′-terminal nucleotide is complementary to the corresponding position onthe reference nucleic acid can be irreversibly terminated whereasextension products whose 3′-terminal nucleotide is not complementary tothe corresponding position on the reference nucleic acid can beproofread and ready to serve as primers for a subsequent extensionreaction which can be detected.

Thus, in one embodiment, the invention provides a method of detectingwithin a first nucleic acid the presence of a nucleotide, comprisinggenerating a set of single-stranded extension products from the firstnucleic acid in the presence of modified nucleotide bases, wherein theextension products incorporate modified nucleotides that limitexonuclease activity to the 3′-terminal nucleotide base, and wherein theextension products have variable lengths; hybridizing the variablelength extension products to a reference nucleic acid; contacting thehybridizing nucleic acids with a first enzyme which can remove the3′-terminal nucleotide of the extension products, and with a secondenzyme which extends those extension products that can be furtherextended in the presence of a selected modified nucleotide which isresistant to further replacement and when incorporated into an extensionproduct inhibits further extension of the extension product, whereinextension products that terminate with a non-modified nucleotide can befurther extended and thereby distinguished from those extension productsthat cannot be further extended; removing the unincorporated selectedmodified nucleotide; extending those extension products that can befurther extended; and distinguishing those extension products that arefurther extended from those extension products that cannot be furtherextended, thereby detecting nucleotide variation in the first nucleicacid. The hybridizing nucleic acids may either be contacted sequentiallywith the first enzyme followed by the second enzyme, or contactedsimultaneously with the first enzyme and the second enzyme.

For a subsequent extension reaction, it may be desirable to remove thechain terminating nucleotide that is resistant to further 3′→5′exonuclease activity from the reaction. This can be accomplished byadding to that reaction mixture an activity that renders the chainterminating nucleotide that is resistant to further 3′→5′ exonucleaseactivity effectively unable to be further added to the single-strandedextension product, such as shrimp alkaline phosphatase which hydrolyzesthe triphosphate on the chain terminating nucleotide, after which theenzyme can be thermally inactivated. Alternatively the template andextension products may be purified from the chain terminating nucleotidethat is resistant to further 3′→5′ exonuclease activity by other methodswell known to those ordinary skill in the art, such as molecular weightor size separation, linking the extension products to a solid support asdiscussed above, and selective degradation of the free nucleotides.

The primer extension reaction in this method may be detected by theincorporation of a label, as discussed above, or indirectly detected,for example, by the release of inorganic pyrophosphate as a result ofpolymerase mediated nucleoside incorporation during DNA synthesis.(Nyren, Anal. Biochem., 167:235-238 (1987)). This embodiment has anadvantage of allowing continuous monitoring of polymerase activitywithin the reaction. This particular reaction can be initiated by addingnucleoside triphosphate bases, D-luciferin, L-luciferin, ATPsulfurylase, luciferase and an oligonucleotide having the same sequenceof a region at or near the 5′ end of the known template and that iscapable of priming an extension reaction. This oligonucleotide can beused to increase the sensitivity of the assay by reverse priming anyfull length extension products that are generated, which is particularlyimportant when a mismatch/mutation occurs near the 5′ end of the knownreference nucleic acid since relatively few nucleotides will be addedprior to reaching the end of the template. For typical luminometricdetection, see Nyren et al. (Anal. Biochem., 244:367-373 (1997)). Theuse of a thermostable luciferase will allow the extension reaction tooccur at higher temperatures and will increase the sensitivity andspecificity of the reaction. (Kaliyama and Nakano, Biosci. Biotechnol.Biochem., 58:1170-1171 (1994)).

Another example of a method for detecting multi-base primer extensionutilizes the Fluorogenic 5′ Nuclease Assay available from Perkin Elmer,Foster City, Calif. and as described by Holland et al., Proc. Natl.Acad. Sci. U.S.A., 88, 7276-7280 (1991). This method involves labeling aprobe, referred to as the TaqMan® probe, with a reporter and quencherdye. The probe can be specific for an internal sequence in theparticular nucleic acid being amplified. As Taq DNA polymerase extendsthe amplification primer it encounters the TaqMan® probe and degrades itwith its 5′ to 3′ exonuclease activity. The dissociation of the reporterfrom the quencher results in an increase in fluorescence. Theapplication of this method to multi-base extension detection requiresonly the synthesis of a TaqMan® probe complementary to the 5′ terminalsequence of the known template. As with bioluminometric detection, thismethod allows continuous monitoring of polymerase activity within thereaction. An additional advantage is the high sensitivity andspecificity since the reaction can be performed at elevated temperaturesin a thermocycling reaction.

In one variation of this method, the step of extending those extensionproducts that can be further extended prior to the detection step can beeliminated where the removal of the unincorporated modified nucleotidecan itself comprise a step that can be detected. For example, where the3′-terminal base is a quenching nucleotide and removal of thatnucleotide allows for detection of the nucleic acid, extension ofextension product to incorporate a detectable moiety is not necessaryfor the subsequent detection step.

Another alternative method for detection comprises incorporating amodified deoxynucleoside triphosphate (dNTP) into the extension product.Examples include radioactive, fluorescent and hapten labeled dNTPs. Afluorescent labeled dNTP, for example, allows direct nonradioactivedetection. The sensitivity of this method, like the bioluminometricdetection, is enhanced by utilizing an oligonucleotide which canhybridize to a region at or near the 5′ end of the known template andthat is capable of priming an extension reaction synthesis from a firstnucleic acid. This method, like the Fluorogenic 5′ Nuclease Assay, hasthe advantage of high sensitivity and specificity since the extensionreaction can be performed at elevated temperatures, such as in athermocycling reaction. This method can add an additional step to removeany unincorporated label, as described above, followed by direct orindirect detection of incorporated label. In one embodiment, solid-phasepurification can be used to capture and purify the extended oligomersfrom any unincorporated fluorescent labeled dNTPs followed byfluorometric detection.

Yet another method of distinguishing those single-stranded extensionproducts that had a 3′-terminal nucleotide that hybridized with thenucleotide at the corresponding position on the reference nucleic acidfrom those that had a 3′-terminal nucleotide that did not hybridize withthe nucleotide at the corresponding position on the reference nucleicacid can comprise separating the products of the method for the presenceof full-length extension products after the proofreading activity hasreplaced the 3′-terminal nucleotide and further extended the extensionproduct by, for example, denaturing gel electrophoresis to visualize thesingle strands, both full-length and those that are not full-length.

In certain applications of the invention it may be desirable to amplifya mutation that compromises a small percentage of the total analyte,such as in early cancer detection where only a few malignant cells maybe present in the total number of cells being analyzed. The selectiveamplification of mutant gene, where the mutation occurs at a known site,such as the K-ras gene, is readily accomplished by the design ofspecific primers to amplify the mutation as described by Stork et al.(Oncogene, 6:857-862 (1991)). However, selective amplification of arandom mutation in a gene among a high percentage of wild type genes isnot possible by standard PCR. In these applications, selectiveamplification of the mutant template may be preferred. Therefore, theextension products from the mutant template are purified from that knowntemplate and hybridized to purified amplified nucleic acid from thesample whose sequence is unknown. The amplified nucleic acid can bepurified from residual primers prior to mixing with the extensionproducts to prevent reamplification of the wild type template. Thehybrid can, for example, be exposed to a thermostable polymerase anddNTPs under thermocycling conditions suitable for polymerization of theextension products. The extension products that were refractory toextension on the known template because of a 3′ terminal mismatch, cannow hybridize to the mutant template from which they were formed and areextended by the polymerase. Preferably, one of the dNTPs is labeled, forexample, with a hapten such as digoxigenin, to allow solid phase captureof the extension products. Solid-phase capture of the hapten labeledextension products followed by standard PCR amplification, with theprimers used for the initial target amplification, preferentiallyamplifies the template containing the mutation. The PCR product can thenbe sequenced by standard methods to specifically identify the preciseposition of the mutation or nucleotide variation.

Thus, in one embodiment, the invention provides a method of selectivelyamplifying a random nucleotide variation in a specific region of anucleic acid molecule present in a sample of nucleic acid moleculescomprising the region, wherein the sample comprises a mixture of nucleicacid molecules comprising the variation, and nucleic acid molecules thatdo not comprise the variation, said method comprising generating a firstset of single-stranded extension products from the sample nucleic acidmolecules in the presence of modified nucleotide bases andchain-terminating nucleotide bases, wherein the extension productsincorporate modified nucleotides that limit exonuclease activity to the3′-terminal nucleotide base, and wherein the extension products havevariable lengths; hybridizing the variable length extension products toa reference nucleic acid that does not have the nucleotide variation;contacting the hybridizing nucleic acids with an enzyme which can removeand replace the 3′-terminal nucleotide of the extension products in thepresence of deoxynucleotide triphosphates, wherein the penultimate3′-nucleotide is resistant to removal from the extension products,whereby extension products containing a penultimate 3′-nucleotide thatdoes not hybridize with the corresponding position on the referencenucleic acid is not replaced with a nucleotide that hybridizes with thecorresponding nucleotide on the reference nucleic acid and therebycannot be further extended; extending those extension products that canbe further extended; separating these extension products from thereference nucleic acid; combining the separated extension products ofstep (e) with a second sample of nucleic acid molecules comprising theregion, wherein the sample comprises a mixture of nucleic acid moleculescomprising the variation, and nucleic acid molecules that do notcomprise the variation; contacting these nucleic acids with a primerextension reaction mixture comprising labeled dNTPS, thus extendingthose extension products that could not be further extended in theprevious extension step; separating the labeled extension products fromthe unlabeled extension products; and amplifying the labeled extensionproducts.

Of course, as in the other methods disclosed herein, the enzyme whichcan remove and replace the 3′-terminal nucleotide base may be any of the“proofreading enzymes” described above, including a combination ofenzymes, such as a first enzyme that removes the 3′-terminal nucleotide(e.g., an exonuclease), and a second enzyme which then replaces that3′-terminal nucleotide (e.g., a polymerase). The hybridizing nucleicacids may either be contacted sequentially with the first enzymefollowed by the second enzyme, or contacted simultaneously with thefirst enzyme and the second enzyme.

The present invention also provides a method of detecting nucleotidevariation within a first nucleic acid, comprising generating a set ofsingle-stranded extension products from the first nucleic acid in thepresence of modified nucleotide bases, and optionally in the presence ofchain-terminating nucleotide bases, wherein the extension productsincorporate modified nucleotides that limit exonuclease activity to the3′-terminal nucleotide base, and wherein the extension products havevariable lengths, hybridizing the variable length extension products toa reference nucleic acid, contacting the hybridizing nucleic acids withan enzyme which can remove and replace the 3′-terminal nucleotide of theextension products in the presence of deoxynucleotide triphosphates,wherein the penultimate 3′-nucleotide is resistant to removal from theextension products, whereby extension products containing a penultimate3′-nucleotide that does not hybridize with the corresponding position onthe reference nucleic acid is not replaced with a nucleotide thathybridizes with the corresponding nucleotide on the reference nucleicacid and thereby cannot be further extended, extending those extensionproducts that can be further extended, distinguishing those extensionproducts that are further extended from those extension products thatcannot be further extended, thereby detecting nucleotide variation inthe first nucleic acid.

This particular method is based on the inability of a proofreadingpolymerase to excise and or extend a mismatched 3′ nucleotide that isresistant to removal by a 3′→5′ exonuclease activity such as athio-modified nucleotide or a borano-modified nucleotide. In thismethod, the set of single-stranded extension products of variable lengthare generated as described above, those extension products arehybridized to a reference nucleic acid, and the hybrid is contacted witha proofreading enzyme, or in an equivalent reaction as discussed above,in the presence of all four deoxynucleotides under conditions suitablefor proofreading and polymerization. (FIGS. 3 and 4) In thosesingle-stranded extension products where the 3′-terminal nucleotidematches the known reference nucleic acid, then the polymerase excisesthe chain-terminating nucleotide or other 3′-terminal nucleotide andreplaces that nucleotide and further extends the extension product.However, if there is a mismatch at the modified nucleotide that isresistant to 3′→5′ exonuclease activity adjacent (i.e. the 3′-terminalnucleotide (FIG. 3) or the penultimate 3′ nucleotide (FIG. 4)) to amatched or mismatched 3′-terminal nucleotide, the proofreading enzymewill not excise that mismatched terminal or penultimate nucleotide andwill be unable to further extend that extension product. The products ofthis reaction can then be analyzed, for example by denaturing gelelectrophoresis, whereby the detection of non-further extended extensionproducts indicates nucleotide variations between the unknown and thereference nucleic acid and further extension of the extension productsindicates that the templates have base complementarity at their 3′-ends,and therefore no nucleotide variations are present at the 3′-end of thefirst nucleic acid relative to the reference nucleic acid.

Thus, in one embodiment, the invention provides a method of detectingnucleotide variation within a first nucleic acid, comprising generatinga set of single-stranded extension products from the first nucleic acidin the presence of modified nucleotide bases and chain-terminatingnucleotide bases, wherein the extension products incorporate modifiednucleotides that limit exonuclease activity to the 3′-terminalnucleotide base, and wherein the extension products have variablelengths; hybridizing the variable length extension products to areference nucleic acid; contacting the hybridizing nucleic acids with afirst enzyme which can remove the 3′-terminal nucleotide of theextension products, and with a second enzyme which extends thoseextension products that can be further extended in the presence ofdeoxynucleotide triphosphates, wherein the penultimate 3′-nucleotide isresistant to removal from the extension products, whereby extensionproducts containing a penultimate 3′-nucleotide that does not hybridizewith the corresponding position on the reference nucleic acid is notreplaced with a nucleotide that hybridizes with the correspondingnucleotide on the reference nucleic acid and thereby cannot be furtherextended; and distinguishing those extension products that are furtherextended from those extension products that cannot be further extended,thereby detecting nucleotide variation in the first nucleic acid. Thehybridizing nucleic acids may either be contacted sequentially with thefirst enzyme followed by the second enzyme, or contacted simultaneouslywith the first enzyme and the second enzyme.

Since the identity of the terminal base is not critical to theembodiment that utilizes a chain-terminating nucleotide, that terminalbase may be removed prior to hybridizing the single-stranded extensionproduct with the reference nucleic acid. Several exonucleases with 3′→5′activity are suitable for this embodiment and are readily available.Some examples are exonuclease I and exonuclease III. Additionally, theremoval of the terminal base obviates the need for a proofreading enzymein the subsequent reaction. Therefore, enzymes which lack the 3′ to 5′exonuclease activity, such as the large fragment of Bst DNA Polymeraseand Taq DNA Polymerase, may be used for extension detection.

Also, since the 3′-terminal nucleotide needs only to be a suitablesubstrate for the extension activities of a polymerase, alternativesequencing methods can be used to generate the set of single-strandedextension products for use in this detection method. Examples includesequencing methods that use thiophosphate or boranophosphate modifiednucleotide as delimiters in a primer extension reaction followed byenzymatic digestion with exonuclease III. (See Labeit et al., DNA, 5:173(1986) and Porter, Nucleic Acids Research 25:1611-1617 (1997)). Theseries of single-stranded extension products of variable lengthgenerated by these processes can be terminated with a thio- orborano-modified nucleotide and are thus well suited for this method.

Other detection methods that may be used in the methods of the inventionare described below.

A labeled dideoxynucleotide may also be rapidly detected by usingMALDI-TOF Mass Spectrophotometry.

In another alternative method of the invention, the primer used forgenerating the extension products is labeled with a ruthenium metalcomplex (ORI-TAG labels from IGEN International) instead of biotin. Abiotin labeled dideoxy nucleotide is then incorporated into theextension products in the final step. These extension products are thencaptured with magnetic beads coated with streptavidin, and the presenceof the ruthenium label is detected. IGEN sells a machine to perform thecapture and detection of the ruthenium label automatically.

Alternatively, the primer used for generating the extension products isunlabeled, and a biotin labeled dideoxy nucleotide is used in the finalstep. A ruthenium labeled probe is then hybridized to the extensionproducts, and this duplex is then captured with magnetic beads coatedwith streptavidin. The ruthenium label may then be detected as describedabove.

The methods of the invention may also be performed in multiplexattaching templates to a specific fluorescent microsphere, thusassociating each template with a unique fluorescent signal, such asFluorescent Microspheres from Luminex. In this manner, an unknown samplemay be simultaneously screened against a number of templates in onereaction vessel. This would be extremely useful when a particular genehas multiple alleles. In methods of the invention where incorporation ofa labeled dideoxt nucleotide is desired (see, e.g., FIG. 1),fluorescently labeled dideoxy nucleotides may be used in a solid-phaseextension reaction.

Of course, the methods of the invention may readily be adapted to amicrotiter plate format using methodology that is well known in the art.One example of such an adaptation is shown in FIG. 5. In this method,the dideoxy terminated sequencing products from the unsequenced templateare mixed with a seqeunced template containing a poly-A capture tail(step 2). A proofreading enzyme or an exonuclease is used to selectivelyremove the terminal dideoxy nucleotide. The extension products are thenextended in the presence of three labeled dideoxy nucleotides and oneunlabeled dideoxy nucleotide that matches the former terminal dideoxynucleotide (step 3). If the terminal dideoxy nucleotide matches thesequenced template, an unlabeled dideoxy will be inserted. If theterminal dideoxy nucleotide does not match the sequenced template, alabeled dideoxy will be inserted. This product is then captured in amicrotiter well coated with a poly T oligonucleotide (Xenopore,Hawthorned, N.J.) The presence of the label is then detected.Preferable, the label is biotin (NEN, Boston, Mass.), and the biotinlabel is detected with a streptavidin alkaline phosphatase conjugatefollowed by luminescent detection in a microplate luminometer.

If greater sensitivity is needed, an alternative to this detectionmethod is to use an anti-digoxigen aequorin conjugate (Sealite Sciences,Norcross, Ga.). In this method, a biotin label is used instead of thepoly-A tail for capture. Digoxigenin labeled dideoxy nucleotides (NEN,Boston, Mass.) can be substituted for the biotin labeled nucleotidesdescribed above, and the extension products captired on a streptavidincoated microtiter plate (Xenopore). This technique has sensitivities inthe 25 attomole range.Nov. 20, 2000

After it has been determined by any of the methods outlined above that acertain nucleic acid comprises a nucleotide variation as compared to thereference nucleic acid, it is possible to specifically identify thenucleotide variation by determining the sequence of the first nucleicacid which has been identified as having a sequence variation, andcomparing that sequence to the sequence of the reference nucleic acid,thereby specifically identifying the nucleotide variation in the firstnucleic acid. The sequence may be determined using any of the standardmethods known in the art. Furthermore, as the first step of the abovemethods generate variable length extension products, it is possible todetermine the sequence of the complement of the first nucleic acid byrunning the variable length extension products on a sequencing gel(i.e., polyacrylamide gel) to generate a sequencing ladder. Of course,in order to do this, a detectable label must be incorporated into theextension products in the initial extension reaction.

Where the method used to determine the presence of a variation insequence results in the production of a primer that is not fullyextended due to a nucleotide variation, as is the case in many of themethods described above, and in FIGS. 1, 3, and 4, that primer may alsobe run on the sequencing gel in order to further confirm the position ofthe sequence variation.

The following example is put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of theclaimed methods, and is intended to be purely exemplary of the inventionand is not intended to limit the scope of what the inventors regard astheir invention. Efforts have been made to ensure accuracy with respectto numbers (e.g., amounts, temperature, etc.) but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in °C. and pressure is at or nearatmospheric.

EXAMPLES

Preparation of Template Molecules

Amplification of K-ras

A 275 base pair region of the K-ras gene exon 1 was amplified by thepolymerase chain reaction (PCR) with the primer K-ras p-A(p-CAGAGAAACCTTTATCTG) (SEQ ID NO: 1) containing a 5′ phosphate andK-ras B (GTACTGGTGGAGTATTT) (SEQ ID NO:2) as disclosed by Stork et al.,Oncogene, 6:857-862 (1991). All primers were synthesized by Oligos Etc.,Inc., Wilsonville, Oreg. The reaction (20 μl) contained 10 mM Tris-HCl,pH 8.3, 50 mM KCl, 2.0 mM MgCl₂, 1 pm/μl Kras A&B primer, 200 μM of eachdNTP 1 ng/μl of K562 genomic DNA (Promega, Madison, Wis.) and 0.025units/μl of Taq DNA polymerase (Perkin Elmer, Foster City, Calif.). Justprior to use the Taq polymerase was mixed with Taq Start antibody(Clontech, Palo Alto, Calif.) according to the manufacturer'sdirections. Thermocycling was done in a GeneAmp 9600 PCR System (PerkinElmer) with the following program: Hold: 95° C. for 5 min, Cycle: 95° C.for 15 sec, 53° C. for 30 sec, 72° C. for 15 sec for 33 cycles, Hold:72° C. for 5 min.

Sequence Determination

The amplified DNA was purified for cycle sequencing using the Wizard PCRPreps DNA Purification System (Promega). It was sequenced in bothdirections by using either the K-ras A or K-ras-B primer. Cyclesequencing and detection was done with the Silver Sequence DNASequencing System (Promega) as described by the manufacturer. Thecycling conditions were: 95° C. for 5 min, Cycle: 95° C. for 30 sec, 50°C. for 30 sec, 72° C. for 60 sec for 60 cycles, Hold: 4° C. forever. Thesequence was confirmed to be the wild type or normal sequence ofcellular c-Ki-ras2 proto-oncogene exon 1 by BLAST analysis (Altschul etal., J. Mol. Biol., 215:403-10 (1990)) against GenBank+EMBL+DDBJ+PDBdatabases at http://www.ncbi.nlm.nih.gov/BLAST.

Generation of Single-stranded Template

The purified PCR product was made single stranded by digestion withlambda exonuclease (Higuchi and Ochman, Nucleic Acids Research, 17:5865(1989)) using the PCR Template Preparation Kit from Pharmacia Biotech,Inc., Piscataway, N.J.

Generation of Dideoxy-terminated Oligomers

Amplification and Sequencing

The same region of the k-ras gene was amplified from genomic DNA fromthe cell line SW480 (American Type Culture Collection, Rockville, Md.)and purified as outlined above. The purified template (40 ng) was mixedwith 10 pm of K-ras b-A primer in 10 μl of 0.7×Sequenase Reaction Buffer(Amersham), heated to 100° C. for 2 min and then immediately cooled inan ice water bath for 5 min. Five μl of Enzyme Mix [1 μl of 0.1 M DTT(Amersham)+2 μl of 5×Sequenase Buffer (Amersham) +2 μl of SequenaseVersion 2.0 (Amersham) diluted 1:8 in Enzyme Dilution Buffer (Amersham)]was added to the ice-cold DNA mixture. This mixture (3.5 μl) was thenadded to a tube at 37° C. containing 2.5 μl of 200 μM of eachalpha-thio-dNTP (Amersham) 1 μM ddATP (Pharmacia). Similarly, this wasrepeated for each of the remaining three ddNTPs. After incubating thesamples at 37° C. for 5 min, 30 μl of 1×TE pH 7.5 (10 mM Tris-Cl pH 7.51 mM EDTA) was added to each tube to stop the reaction.

Purification of Extension Products from Template

Streptavidin-coated magnetic beads (Dynabeads M-280, Dynal Inc., LakeSuccess, N.Y.) were used for solid-phase capture of extension products.Prior to use, Dynabeads were washed in 2×B&W buffer (10 mM Tris-HCl, pH7.5, 1 mM EDTA, 2 M NaCl) according the manufacturer's directions. Foreach completed dideoxy sequencing reaction, 40 μl of 1.25 μg/μlstreptavidin-coated magnetic beads in 2×B&W buffer was added to eachreaction. After incubating for 15 minutes at room temperature withintermittent vortexing the beads were captured and washed with 40 μl of2×B&W buffer. Melting of the DNA duplex and strand separation wereperformed according to the manufacturer's directions.

Generation of Alpha-thio Terminated Oligomers from a Mutant and WildType Template

The same region of the k-ras gene was amplified from genomic DNA fromthe both the K562 and SW480 (American Type Culture Collection,Rockville, Md.) cell line and purified as outlined above. The purifiedtemplate (240 ng) was mixed with 60 pm of K-ras b-A primer in 60 μl of0.07×Sequenase Reaction Buffer, dispensed in three 20 μl aliquots,heated to 100° C. for 3 min and then immediately cooled in an ice waterbath for 5 min. Ten μl of Enzyme Mix (1 μl of 0.1 M DTT+2 μl of5×Sequenase Buffer+2 μl of Sequenase Version 2.0 diluted 1:8 in EnzymeDilution Buffer) was added to the ice-cold DNA mixture. This mixture (28μl) was then added to a tube at 37° C. containing 20 μl of 26 μMalpha-thio-dATP 54 μM dATP 80 μM of the remaining three dNTPs. Afterincubating the samples at 37° C. for 5 min the reaction was inactivatedby heating at 75° C. for 10 minutes. Sixteen μl of a solution containing20 U/μl Exonuclease III (Promega) in 7×Exonuclease III Buffer (Promega)was added to each inactivated extension reaction. The reactions wereincubated at 37° C. for 30 minutes and then heat inactivated at 70° C.for 10 minutes.

Extension Detection of Nucleotide Variations

Non-extension Detection

Two hybridization reactions were set up for SW480 and K562 alpha-thioterminated oligomers, one containing ss k-ras template DNA from K562 andone without template DNA. The template containing hybridization reactioncontained the following: 36 μl of alpha-thio terminated oligomers fromeither K562 or SW480, 100 ng of ss k-ras template from K562, 60 μl of2×B&W buffer (10 mM Tris-HCl, pH 7.5, 1.0 mM EDTA, 2 M NaCl) andmolecular biology grade (MBG) water (Sigma, W-4502, St. Louis, Mo.) to afinal volume of 120 μl. A parallel reaction was setup without templateDNA. The reactions were heated at 99.9° C. for 2 min and cooled 1°C./min to 58° C. The reaction was transferred to a Microcon YM-30centrifugal filter device and spun 5 minutes at 7200×g. The retentatewas mixed with 450 μl of MBG water and reconcentrated two times. Theretentate was adjusted to a final volume of 12 μl with MBG water. Six μlof the retenatete was mixed with 4 μl extension master mix: 25 mMTris-HCl, pH 8.3, 125 mM KCl, 5.0 mM MgCl₂, 500 μM of each dNTP and0.125 units/μl of Taq DNA polymerase (Perkin Elmer). Just prior to usethe Taq polymerase was mixed with Taq Start antibody (Clontech, PaloAlto, Calif.) according to the manufacturer's directions. The reactionswere thermocycled as follows: Hold: 95° C. for 5 min, Cycle: 95° C. for15 sec then 94° C. for 15 sec with a 2° C. decrease/cycle for 14 cycles,Cycle: 95° C. for 15 sec, 68° C. for 15 sec with a 2° C. decrease/cycle,72° C. for 15 sec for 19 cycles. Upon completion, 4 μl of stop solution(95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanolFF) was added to each reaction. Three μl of the supernatant was loadedon a 0.4 mm×20 cm×34 cm 6% denaturing polyacrylamide gel for analysis.The gel was preheated at 40 W for 30 minutes before loading the samplesand running at 40 w for 50 min. The Phototope-Star Detection Kit (NewEngland Biolabs) was used for chemiluminescent detection of thebiotinylated extension products as described by the manufacturer.

Single Base Extension Detection

Six μl of single-base extension master mix containing: 1×ThermoPolReaction Buffer (New England Biolabs, Inc., Beverly, Mass.) 2.0 mMMgSO₄, 200 μM of an unlabeled ddNTP that matches the terminaldideoxynucleotide, 200 μM of the 3 remaining ddNTPs labeled with ³³P(Amersham), 1 ng/μl of single-stranded template DNA and 0.01 units/μl ofVent DNA polymerase (New England Biolabs, Inc.) was used to suspend thesolid-phase extension products. Thermocycling was as follows: Hold: 95°C. for 5 min, Cycle: 95° C. for 15 sec then 94° C. for 15 sec with a 2°C. decrease/cycle for 14 cycles, Cycle: 95° C. for 15 sec, 68° C. for 15sec with a 2° C. decrease/cycle, 72° C. for 15 sec for 19 cycles, Hold:4° C.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

Although the present process has been described with reference tospecific details of certain embodiments thereof, it is not intended thatsuch details should be regarded as limitations upon the scope of theinvention except as and to the extent that they are included in theaccompanying claims.

2 1 18 DNA Artificial Sequence Oligonucleotide, Single-StrandedAmplification Primer 1 cagagaaacc tttatctg 18 2 17 DNA ArtificialSequence Oligonucleotide, Single-Stranded Amplification Primer 2gtactggtgg agtattt 17

What is claimed is:
 1. A method of detecting a nucleotide variationwithin a first nucleic acid, comprising: a) generating a set ofsingle-stranded extension products from the first nucleic acid in thepresence of modified nucleotide bases, wherein the extension productsincorporate modified nucleotides and thereby limit exonuclease activityto the 3′-terminal nucleotide base, and wherein the extension productshave variable lengths; b) hybridizing the variable length extensionproducts to a reference nucleic acid; c) contacting the hybridizingnucleic acids with a first enzyme which can remove the 3′-terminalnucleotide of the extension products, and with a second enzyme whichextends those extension products that can be further extended in thepresence of selected labeled nucleotides, wherein extension productsthat terminate with a 3′-nucleotide that does not hybridize with thecorresponding position on the reference nucleic acid are replaced with anucleotide that hybridizes with the corresponding nucleotide on thereference nucleic acid and wherein those extension products that had anon-hybridizing nucleotide at the 3′-terminus can now be distinguishedfrom those extension products that had a hybridizing nucleotide at the3′-terminus; and d) distinguishing those extension products that had anon-hybridizing nucleotide at their 3′-terminus from those extensionproducts that had a hybridizing nucleotide at their 3′-terminus, therebydetecting nucleotide variation in the first nucleic acid.
 2. The methodof claim 1, wherein the modified nucleotides comprise thio-modifieddeoxynucleotides.
 3. The method of claim 1, wherein the modifiednucleotides comprise borano-modified deoxynucleotides.
 4. The method ofclaim 1, wherein the first enzyme of step (c) is a 3′ to 5′ exonuclease,and the second enzyme of step (c) is a DNA polymerase.
 5. The method ofclaim 1, wherein in step (c), the hybridizing nucleic acids arecontacted sequentially with the first enzyme followed by the secondenzyme.
 6. The method of claim 1, wherein in step (c), the hybridizingnucleic acids are contacted simultaneously with the first enzyme and thesecond enzyme.
 7. The method of claim 1, wherein the extension productsare modified.
 8. The method of claim 7, wherein the modificationcomprises linking biotin to the extension product.
 9. The method ofclaim 7, wherein the modification comprises linking a hapten to theextension product.
 10. The method of claim 7, further comprisingisolating the extension products of step (a) by binding them to a solidsupport.
 11. The method of claim 8, wherein the extension products arelabeled with biotin and are isolated by binding them to a solid supportcoated with streptavidin.
 12. The method of claim 1, wherein step (c)further comprises performing the contacting step in the presence of theselected nucleotide base in the form of a second label-labeleddideoxynucleoside triphosphate, thereby providing a positive control forfunctioning of the enzyme.
 13. The method of claim 1, wherein theextension products are variable length by incorporation ofchain-terminating nucleotides into the extension products.
 14. Themethod of claim 1, wherein the extension products are variable length bypartial exonuclease digestion of the extension products.
 15. The methodof claim 1, comprising the additional step of determining the sequenceof the first nucleic acid which has been identified as having a sequencevariation and comparing that sequence to the sequence of the referencenucleic acid, thereby specifically identifying the nucleotide variationin the first nucleic acid.
 16. A method of detecting a nucleotidevariation within a first nucleic acid and specifically identifying thenucleotide variation, comprising: a) generating a set of single-strandedextension products from the first nucleic acid in the presence ofmodified nucleotide bases, wherein the extension products incorporatemodified nucleotides and thereby limit exonuclease activity to the3′-terminal nucleotide base, and wherein the extension products havevariable lengths; b) hybridizing the variable length extension productsto a reference nucleic acid; c) contacting the hybridizing nucleic acidswith an enzyme which can remove and replace the 3′-terminal nucleotideof the extension products in the presence of selected labelednucleotides, wherein extension products that terminate with a3′-nucleotide that does not hybridize with the corresponding position onthe reference nucleic acid are replaced with a nucleotide thathybridizes with the corresponding nucleotide on the reference nucleicacid and wherein those extension products that had a non-hybridizingnucleotide at the 3′-terminus can now be distinguished from thoseextension products that had a hybridizing nucleotide at the 3′-terminus;d) distinguishing those extension products that had a non-hybridizingnucleotide at their 3′-terminus from those extension products that had ahybridizing nucleotide at their 3′-terminus, thereby detectingnucleotide variation in the first nucleic acid; and e) determining thesequence of the first nucleic acid which has been identified as having asequence variation and comparing that sequence to the sequence of thereference nucleic acid, thereby specifically identifying the nucleotidevariation.
 17. A method of detecting within a first nucleic acid thepresence of a nucleotide, comprising: a) generating a set ofsingle-stranded extension products from the first nucleic acid in thepresence of modified nucleotide bases, wherein the extension productsincorporate modified nucleotides that limit exonuclease activity to the3′-terminal nucleotide base, and wherein the extension products havevariable lengths; b) hybridizing the variable length extension productsto a reference nucleic acid; c) contacting the hybridizing nucleic acidswith a first enzyme which can remove the 3′-terminal nucleotide of theextension products, and with a second enzyme which extends thoseextension products that can be further extended in the presence of aselected modified nucleotide which is resistant to further replacementand when incorporated into an extension product inhibits furtherextension of the extension product, wherein extension products thatterminate with a non-modified nucleotide can be further extended andthereby distinguished from those extension products that cannot befurther extended; d) removing the unincorporated selected modifiednucleotide; e) extending those extension products that can be furtherextended; and f) distinguishing those extension products that arefurther extended from those extension products that cannot be furtherextended, thereby detecting nucleotide variation in the first nucleicacid.
 18. The method of claim 17, wherein the selected modifiednucleotide comprises a thio-modified dideoxynucleotide triphosphate. 19.The method of claim 17, wherein the selected modified nucleotidecomprises a borano-modified dideoxynucleotide triphosphate.
 20. Themethod of claim 17, wherein step (d) further comprises adding the enzymeshrimp alkaline phosphatase under conditions suitable for activity ofthe enzyme followed by inactivating the enzyme.
 21. The method of claim17, wherein in step (c), the hybridizing nucleic acids are contactedsequentially with the first enzyme followed by the second enzyme. 22.The method of claim 17, wherein in step (c), the hybridizing nucleicacids are contacted simultaneously with the first enzyme and the secondenzyme.
 23. The method of claim 17, wherein the extension products aremodified.
 24. The method of claim 23, wherein the modification compriseslinking biotin to the extension product.
 25. The method of claim 23,wherein the modification comprises linking a hapten to the extensionproduct.
 26. The method of claim 17, further comprising isolating theextension products of step (a) by binding them to a solid support. 27.The method of claim 24, wherein the extension products are labeled withbiotin and are isolated by binding them to a solid support coated withstreptavidin.
 28. The method of claim 17, wherein the extension productsare variable length by incorporation of chain-terminating nucleotidesinto the extension products.
 29. The method of claim 17, wherein theextension products are variable length by partial exonuclease digestionof the extension products.
 30. The method of claim 17, comprising theadditional step of determining the sequence of the first nucleic acidwhich has been identified as having a sequence variation and comparingthat sequence to the sequence of the reference nucleic acid, therebyspecifically identifying the nucleotide variation in the first nucleicacid.
 31. A method of detecting within a first nucleic acid the presenceof a nucleotide variation and specifically identifying the nucleotidevariation, comprising: a) generating a set of single-stranded extensionproducts from the first nucleic acid in the presence of modifiednucleotide bases, wherein the extension products incorporate modifiednucleotides that limit exonuclease activity to the 3′-terminalnucleotide base, and wherein the extension products have variablelengths; b) hybridizing the variable length extension products to areference nucleic acid; c) contacting the hybridizing nucleic acids withan enzyme which can remove and replace the 3′-terminal nucleotide of theextension products in the presence of a selected modified nucleotidewhich is resistant to further replacement and when incorporated into anextension product inhibits further extension of the extension product,wherein extension products that terminate with a non-modified nucleotidecan be further extended and thereby distinguished from those extensionproducts that cannot be further extended; d) removing the unincorporatedselected modified nucleotide; e) extending those extension products thatcan be further extended; f) distinguishing those extension products thatare further extended from those extension products that cannot befurther extended, thereby detecting nucleotide variation in the firstnucleic acid; and g) determining the sequence of the first nucleic acidwhich has been identified as having a sequence variation and comparingthat sequence to the sequence of the reference nucleic acid, therebyspecifically identifying the nucleotide variation.
 32. A method ofdetecting nucleotide variation within a first nucleic acid, comprising:a) generating a set of single-stranded extension products from the firstnucleic acid in the presence of modified nucleotide bases andchain-terminating nucleotide bases, wherein the extension productsincorporate modified nucleotides that limit exonuclease activity to the3′-terminal nucleotide base, and wherein the extension products havevariable lengths; b) hybridizing the variable length extension productsto a reference nucleic acid; c) contacting the hybridizing nucleic acidswith a first enzyme which can remove the 3′-terminal nucleotide of theextension products, and with a second enzyme which extends thoseextension products that can be further extended in the presence ofdeoxynucleotide triphosphates, wherein the penultimate 3′-nucleotide isresistant to removal from the extension products, whereby extensionproducts containing a penultimate 3′-nucleotide that does not hybridizewith the corresponding position on the reference nucleic acid is notreplaced with a nucleotide that hybridizes with the correspondingnucleotide on the reference nucleic acid and thereby cannot be furtherextended; and d) distinguishing those extension products that arefurther extended from those extension products that cannot be furtherextended, thereby detecting nucleotide variation in the first nucleicacid.
 33. The method of claim 32, wherein in step (c), the hybridizingnucleic acids are contacted sequentially with the first enzyme followedby the second enzyme.
 34. The method of claim 32, wherein in step (c),the hybridizing nucleic acids are contacted simultaneously with thefirst enzyme and the second enzyme.
 35. The method of claim 32, whereinthe single stranded extension products are labeled with a detectablelabel and the distinguishing step (d) comprises performing gelelectrophoresis of the reaction from step (c), wherein theelectrophoresis is of sufficient resolution to distinguish between alabeled extension product that is not further extended and a labeledextension product that is further extended.
 36. The method of claim 32,wherein the modified nucleotides comprise thio-modifieddeoxynucleotides.
 37. The method of claim 32, wherein the modifiednucleotides comprise borano-modified deoxynucleotides.
 38. The method ofclaim 32, wherein the extension products are modified.
 39. The method ofclaim 38, wherein the modification comprises linking biotin to theextension product.
 40. The method of claim 39, wherein the modificationcomprises linking a hapten to the extension product.
 41. The method ofclaim 32, further comprising isolating the extension products of step(a) by binding them to a solid support.
 42. The method of claim 39,wherein the extension products are labeled with biotin and are isolatedby binding them to a solid support coated with streptavidin.
 43. Themethod of claim 32, comprising the additional step of determining thesequence of the first nucleic acid which has been identified as having asequence variation and comparing that sequence to the sequence of thereference nucleic acid, thereby specifically identifying the nucleotidevariation in the first nucleic acid.
 44. A method of detectingnucleotide variation within a first nucleic acid, and specificallyidentifying the nucleotide variation, comprising: a) generating a set ofsingle-stranded extension products from the first nucleic acid in thepresence of modified nucleotide bases and chain-terminating nucleotidebases, wherein the extension products incorporate modified nucleotidesthat limit exonuclease activity to the 3′-terminal nucleotide base, andwherein the extension products have variable lengths; b) hybridizing thevariable length extension products to a reference nucleic acid; c)contacting the hybridizing nucleic acids with an enzyme which can removeand replace the 3′-terminal nucleotide of the extension products in thepresence of deoxynucleotide triphosphates, wherein the penultimate3′-nucleotide is resistant to removal from the extension products,whereby extension products containing a penultimate 3′-nucleotide thatdoes not hybridize with the corresponding position on the referencenucleic acid is not replaced with a nucleotide that hybridizes with thecorresponding nucleotide on the reference nucleic acid and therebycannot be further extended; d) extending those extension products thatcan be further extended; e) distinguishing those extension products thatare further extended from those extension products that cannot befurther extended, thereby detecting nucleotide variation in the firstnucleic acid; and f) determining the sequence of the first nucleic acidwhich has been identified as having a sequence variation and comparingthat sequence to the sequence of the reference nucleic acid, therebyspecifically identifying the nucleotide variation.
 45. A method ofdetecting within a first nucleic acid the presence of a nucleotidevariation and specifically identifying the nucleotide variation,comprising: a) generating a set of single-stranded extension productsfrom the first nucleic acid in the presence of modified nucleotidebases, wherein the extension products incorporate modified nucleotidesthat inhibit exonuclease activity at the 3′-terminal nucleotide base,and wherein the extension products have variable lengths; b) hybridizingthe variable length extension products to a reference nucleic acid; c)contacting the hybridizing nucleic acids with an enzyme which, in thepresence of deoxynucleotide triphosphates, can further extend thoseextension products that have a 3′-terminal nucleotide that hybridizeswith the corresponding position on the reference nucleic acid andwhereby extension products containing a 3′-terminal nucleotide that doesnot hybridize with the corresponding position on the reference nucleicacid are not further extended, and wherein an extension product thatcannot be further extended is generated from a first nucleic acid thatcomprises a nucleotide variation; d) extending those extension productsthat can be further extended; e) distinguishing those extension productsthat are further extended from those extension products that cannot befurther extended, thereby detecting nucleotide variation in the firstnucleic acid; and f) determining the sequence of the first nucleic acidwhich has been identified as having a sequence variation and comparingthat sequence to the sequence of the reference nucleic acid, therebyspecifically identifying the nucleotide variation.
 46. The method ofclaim 45, wherein the extension products are variable length by partialexonuclease digestion of the extension products.
 47. A method ofselectively amplifying a random nucleotide variation in a specificregion of a nucleic acid molecule present in a sample of nucleic acidmolecules comprising the region, wherein the sample comprises a mixtureof nucleic acid molecules comprising the variation, and nucleic acidmolecules that do not comprise the variation, said method comprising: a)generating a first set of single-stranded extension products from thesample nucleic acid molecules in the presence of modified nucleotidebases and chain-terminating nucleotide bases, wherein the extensionproducts incorporate modified nucleotides that limit exonucleaseactivity to the 3′-terminal nucleotide base, and wherein the extensionproducts have variable lengths; b) hybridizing the variable lengthextension products of step (a) to a reference nucleic acid that does nothave the nucleotide variation; c) contacting the hybridizing nucleicacids with a first enzyme which can remove the 3′-terminal nucleotide ofthe extension products, and with a second enzyme which extends thoseextension products that can be further extended in the presence ofdeoxynucleotide triphosphates, wherein the penultimate 3′-nucleotide isresistant to removal from the extension products, whereby extensionproducts containing a penultimate 3′-nucleotide that does not hybridizewith the corresponding position on the reference nucleic acid is notreplaced with a nucleotide that hybridizes with the correspondingnucleotide on the reference nucleic acid and thereby cannot be furtherextended; d) separating the extension products of step (d) from thereference nucleic acid; e) combining the separated extension products ofstep (e) with a second sample of nucleic acid molecules comprising theregion, wherein the sample comprises a mixture of nucleic acid moleculescomprising the variation, and nucleic acid molecules that do notcomprise the variation; f) contacting the nucleic acids of step (f) witha primer extension reaction mixture comprising labeled dNTPS, thusextending those extension products that could not be further extended instep (d); (g) separating the labeled extension products from theunlabeled extension products; and (h) amplifying the labeled extensionproducts.
 48. The method of claim 47, wherein in step (c), thehybridizing nucleic acids are contacted sequentially with the firstenzyme followed by the second enzyme.
 49. The method of claim 47,wherein in step (c), the hybridizing nucleic acids are contactedsimultaneously with the first enzyme and the second enzyme.
 50. Themethod of claim 47, further comprising sequencing the labeled extensionproducts of step (i), and comparing that sequence to the sequence of thereference nucleic acid, thus specifically identify the nucleotidevariation.
 51. A method of selectively amplifying a random nucleotidevariation in a specific region of a nucleic acid molecule present in asample of nucleic acid molecules comprising the region, wherein thesample comprises a mixture of nucleic acid molecules comprising thevariation, and nucleic acid molecules that do not comprise thevariation, said method comprising: a) generating a first set ofsingle-stranded extension products from the sample nucleic acidmolecules in the presence of modified nucleotide bases andchain-terminating nucleotide bases, wherein the extension productsincorporate modified nucleotides that limit exonuclease activity to the3′-terminal nucleotide base, and wherein the extension products havevariable lengths; b) hybridizing the variable length extension productsof step (a) to a reference nucleic acid that does not have thenucleotide variation; c) contacting the hybridizing nucleic acids withan enzyme which can remove and replace the 3′-terminal nucleotide of theextension products in the presence of deoxynucleotide triphosphates,wherein the penultimate 3′-nucleotide is resistant to removal from theextension products, whereby extension products containing a penultimate3′-nucleotide that does not hybridize with the corresponding position onthe reference nucleic acid is not replaced with a nucleotide thathybridizes with the corresponding nucleotide on the reference nucleicacid and thereby cannot be further extended; d) extending thoseextension products that can be further extended; e) separating theextension products of step (d) from the reference nucleic acid; f)combining the separated extension products of step (e) with a secondsample of nucleic acid molecules comprising the region, wherein thesample comprises a mixture of nucleic acid molecules comprising thevariation, and nucleic acid molecules that do not comprise thevariation; g) contacting the nucleic acids of step (f) with a primerextension reaction mixture comprising labeled dNTPS, thus extendingthose extension products that could not be further extended in step (d);(h) separating the labeled extension products from the unlabeledextension products; and (i) amplifying the labeled extension products.52. The method of claim 51, further comprising sequencing the labeledextension products of step (i), and comparing that sequence to thesequence of the reference nucleic acid, thus specifically identify thenucleotide variation.